Spatial transcriptomics implicates impaired BMP signaling in NF1 fracture pseudarthrosis in murine and patient tissues

The Neurofibromatosis Type 1 (NF1) RASopathy is associated with persistent fibrotic nonunions (pseudarthrosis) in human and mouse skeletal tissue. Here, we first performed spatial transcriptomics to define the molecular signatures across normal endochondral healing following fracture in mice. Within the control fracture callus, we observed spatially restricted activation of morphogenetic pathways, such as TGF-β, WNT, and BMP. To investigate the molecular mechanisms contributing to Nf1-deficient delayed fracture healing, we performed spatial transcriptomic analysis on a Postn-cre;Nf1flox/- (Nf1Postn) fracture callus. Transcriptional analyses, subsequently confirmed through p-SMAD1/5/8 immunohistochemistry, demonstrated a lack of BMP pathway induction in Nf1Postn mice. To further inform the human disease, we performed spatial transcriptomic analysis of fracture pseudarthrosis tissue from a NF1 patient. Analyses detected increased MAPK signaling at the fibrocartilaginous-osseus junction. Similar to the Nf1Postn fracture, BMP pathway activation was absent within the pseudarthrosis tissue. Our results demonstrate the feasibility to delineate the molecular and tissue-specific heterogeneity inherent in complex regenerative processes, such as fracture healing, and to reconstruct phase transitions representing endochondral bone formation in vivo. Furthermore, our results provide in situ molecular evidence of impaired BMP signaling underlying NF1 pseudarthrosis, potentially informing the clinical relevance of off-label BMP2 as a therapeutic intervention.